Application of ZmCML11 to improve low temperature germination ability of corn seeds

Abstract

The present application relates to the technical field of low-temperature germination of corn, and particularly relates to an application of a corn gene. The present application excavates a calmodulin protein ZmCML11 from a low-temperature-resistant germplasm resource unique in China. As a kind of calmodulin protein, the ZmCML11 interacts with a Zmzim23 which is an important inhibitor of a jasmonic acid signal pathway, and then influences the jasmonic acid signal pathway, and plays an important role in the germination of corn at low temperature. The application of the ZmCML11 in corn planting can improve the corn yield level and guarantee food security.

Description

Technical Field

[0001] This invention relates to the field of low-temperature germination technology for maize, and in particular to the application of the maize ZmCML11 gene. Background Technology

[0002] Currently, maize (Zea mays L.) has become the world's highest-yielding crop, playing a crucial role in ensuring global food security. As my country's largest grain crop, maize occupies a strategic position in the agricultural economy. Primarily used as animal feed, industrial processed products, and a staple food, maize is a key pillar for maintaining livestock development, bio-based material manufacturing, and food reserve security. In recent years, the average annual growth rate of total maize consumption in my country has exceeded the growth rate of production, creating a supply-demand gap that urgently requires breakthroughs in yield per unit area. However, with declining arable land quality and decreasing acreage, and no significant increase in maize planting area, improving maize yield per unit area is a crucial measure for ensuring my country's food security.

[0003] Originating in tropical and subtropical regions, maize is sensitive to low temperatures. In the maize-producing areas of northern my country, low-temperature chilling injury is one of the major meteorological disasters. Low-temperature chilling injury in maize typically occurs during the early spring sowing and seedling emergence stage, when seeds are germinating. At this time, low temperatures slow down the germination rate, preventing normal emergence or hindering growth after emergence, ultimately leading to missing seedlings, broken rows, and reduced yield. Low-temperature stress during the seed emergence stage not only delays germination time and reduces the germination rate but also increases the likelihood of soil pathogens infecting seeds, severely impacting seedling survival rate and hindering high and stable yields. Statistics show that low-temperature chilling injury typically causes a 5%–15% yield reduction, while severe low-temperature chilling injury can reduce yields by 15%–25%. Therefore, improving seed germination ability under low temperatures is crucial for maize production.

[0004] In plants, Ca 2+ It is an important second messenger, and its signal formation and transmission are key steps in plant response to low-temperature stress. Changing calcium signals are chemical signals and must be decoded before they can be sensed by plant cells. In plants, Ca... 2+ The decoding process of the signal is accomplished by calcium-binding proteins. Calmodulin-like proteins (CMLs) are plant-specific calcium-binding proteins, with multiple members found in different plants. Their role in the low-temperature germination process of maize seeds has not yet been reported. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an application of the maize ZmCML11 gene, which is overexpressed in maize plants to improve the cold resistance of maize.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides for the use of the ZmCML11 gene or the protein it encodes in any of the following:

[0008] (i) Prediction of maize traits;

[0009] (ii) Maize breeding;

[0010] (iii) Maize genetic improvement;

[0011] The nucleotide sequence of the ZmCML11 gene has:

[0012] (1) The nucleotide sequence as shown in SEQ ID NO: 1; or

[0013] (2) A nucleotide sequence obtained by substitution, deletion or addition of one or more bases to the nucleotide sequence shown in (1), and whose function is the same as or similar to that of (1); or

[0014] (3) A nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the nucleotide sequence shown in (1) or (2);

[0015] The amino acid sequence of the protein has:

[0016] (4) The amino acid sequence as shown in SEQ ID NO: 2; or

[0017] (5) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (4), and whose function is the same as or similar to that of (4); or

[0018] (6) An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (4) or (5).

[0019] The plurality can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0020] In some specific embodiments of the present invention, the maize trait prediction described above includes:

[0021] (i) the ability to predict low-temperature germination in maize; and / or

[0022] (ii) Predicting the cold resistance of maize.

[0023] In some specific embodiments of the present invention, the maize breeding described above includes:

[0024] (iii) Screening for maize varieties with low-temperature germination ability; and / or

[0025] (iv) Select corn varieties with higher cold resistance;

[0026] In some specific embodiments of the present invention, the above-described application of maize genetic improvement includes:

[0027] (v) The ability to induce low-temperature germination in maize; and / or

[0028] (vi) Improve the cold resistance of corn.

[0029] The present invention also provides a method for predicting maize traits, which includes: prediction based on the ZmCML11 gene or its encoded protein as described in the above applications.

[0030] In some specific embodiments of the present invention, the above-mentioned maize trait prediction method includes: a higher expression level of the ZmCML11 gene or its encoded protein results in a higher low-temperature germination rate and / or higher cold resistance in maize.

[0031] The present invention also provides a method for breeding maize, which includes: breeding based on the ZmCML11 gene or the protein encoded by it described in the above applications.

[0032] In some specific embodiments of the present invention, the above-mentioned maize breeding method includes: screening maize according to the expression level of the ZmCML11 gene or its encoded protein to obtain maize with higher low-temperature germination rate and / or higher cold resistance.

[0033] The present invention also provides a method for genetic improvement of maize, which includes: genetic improvement based on the ZmCML11 gene or the protein encoded by it described in the above applications.

[0034] In some specific embodiments of the present invention, the above-mentioned maize genetic improvement method includes: overexpressing the ZmCML11 gene or its encoded protein as described in the above application to obtain maize with higher low-temperature germination rate and / or higher cold resistance.

[0035] In some specific embodiments of the present invention, in the above-mentioned maize genetic improvement method, the ZmCML11 gene or its encoded protein interacts with the Zmzim23 protein to affect the jasmonic acid signaling pathway, thereby regulating the low-temperature germination and / or cold resistance of maize seeds.

[0036] In some specific embodiments of the present invention, the above-mentioned method for genetic improvement of maize includes:

[0037] The nucleic acid molecules of the ZmCML11 gene were linked to the backbone to obtain the expression vector;

[0038] Transform Agrobacterium using the expression vector;

[0039] The embryos of the maize inbred line CAL were infected with Agrobacterium, induced, co-cultured, screened, differentiated, rooted, and transplanted to obtain positive ZmCML11 transgenic plants.

[0040] In some specific embodiments of the present invention, the above-mentioned maize genetic improvement method further includes the step of identifying ZmCML11 transgenic positive plants.

[0041] This invention also provides biomaterials for maize trait prediction, maize breeding, or maize genetic improvement, comprising:

[0042] (i) the amplification primers for the ZmCML11 gene described in the above applications; and / or

[0043] (ii) Capture antibodies against the proteins described in the above applications; and / or

[0044] (iii) Expression cassettes, expression vectors and / or recombinant host cells that express the ZmCML11 gene or the protein encoded by it as described in the above applications, wherein the host cells do not include cells capable of developing into complete plants.

[0045] In some specific embodiments of the present invention, the amplification primers of the above-mentioned biological materials are not limited to amplifying the full length, part, or sufficient to prove the presence of the ZmCML11 gene in the nucleic acid molecules of the sample to be tested.

[0046] The present invention also provides reagents, kits, or devices comprising the aforementioned biological materials.

[0047] In some specific embodiments of the present invention, the above-mentioned reagents, kits or devices are used for purposes including but not limited to maize trait prediction, maize breeding or maize genetic improvement.

[0048] The present invention has the following beneficial effects:

[0049] To address chilling injury during maize germination, a calmodulin-like protein, ZmCML11, was identified from low-temperature tolerant germplasm resources unique to my country. As a calmodulin protein, research shows that ZmCML11 interacts with Zmzim23, an important repressor of the jasmonic acid signaling pathway, thereby affecting the jasmonic acid signaling pathway and playing a crucial role in maize germination under low temperatures. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art are briefly introduced below.

[0051] Figure 1 The expression patterns of ZmCML11 are shown, where (a) shows the difference in ZmCML11 expression in different germination states of Y1518 and 220 seeds, (b) shows the expression patterns of ZmCML11 during the germination process of 220 and Y1518 seeds, and (c) shows the subcellular localization of ZmCML11 protein in maize protoplasts.

[0052] Figure 2 Phenotypic identification of ZmCML11 knockout line is shown, where (a) shows the editing method of cristr#1 and cristr#2, (b) shows the room temperature germination phenotype of WT and cristr#1, cristr#2, (c) shows the room temperature germination GR of WT and cristr#1 and cristr#2, (d) shows the room temperature germination GI of WT and cristr#1 and cristr#2, (e) shows the room temperature germination MGT of WT and cristr#1 and cristr#2, (f) shows the low temperature germination phenotype of WT and cristr#1 and cristr#2, (g) shows the low temperature germination GR of WT and cristr#1 and cristr#2, (h) shows the low temperature germination GI of WT and cristr#1 and cristr#2, and (i) shows the low temperature germination MGT of WT and cristr#1 and cristr#2.

[0053] Figure 3 The ZmCML11 overexpression phenotypes were identified as follows: (a) shows the overexpression fold of OE#1 and OE#2; (b) shows the room temperature germination phenotype of WT and OE#1 and OE#2; (c) shows the room temperature germination GR of WT and OE#1 and OE#2; (d) shows the room temperature germination GI of WT and OE#1 and OE#2; (e) shows the room temperature germination MGT of WT and OE#1 and OE#2; (f) shows the low temperature germination phenotype of WT and OE#1 and OE#2; (g) shows the low temperature germination GR of WT and OE#1 and OE#2; (h) shows the low temperature germination GI of WT and OE#1 and OE#2; and (i) shows the low temperature germination MGT of WT and OE#1 and OE#2.

[0054] Figure 4 The interaction between ZmCML11 and Zmzim23 proteins was verified, including (a) a yeast two-hybrid assay, (b) a luciferase complementation assay, and (c) a bimolecular fluorescence complementation assay.

[0055] Figure 5 The expression differences of marker genes in the jasmonic acid signaling pathway were verified. Among them, (a) showed the expression differences of lox10 and lox11 genes in WT and cristr#1 and cristr#2, (b) showed the expression differences of aos4 and aos3 genes in WT and cristr#1 and cristr#2, (c) showed the expression differences of aoc1 and aoc2 genes in WT and cristr#1 and cristr#2, and (d) showed the expression differences of opr7 and opr8 genes in WT and cristr#1 and cristr#2. Detailed Implementation

[0056] This invention discloses an application of the maize ZmCML11 gene. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0057] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0058] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0059] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0060] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0061] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0062] The present invention relates to an overexpression of the maize ZmCML11 gene in maize plants to improve the low-temperature germination tolerance of maize seeds. Specifically, the nucleotide sequence of the maize ZmCML11 gene is shown in SEQ ID NO. 1, and the amino acid sequence of the maize ZmCML11 gene is shown in SEQ ID NO. 2.

[0063] Preferably, the method for overexpressing the maize ZmCML11 gene in maize plants is as follows:

[0064] Cloning of S1 and ZmCML11 genes:

[0065] S101. Select the maize B73 inbred line as the experimental material, extract RNA from germinated embryos after 24 h of germination, and reverse transcribe it into cDNA;

[0066] S102. Using the cDNA obtained in S101 as a template, design adapter primers ZmCML11-F and ZmCML11-R as primers for PCR amplification to obtain PCR products.

[0067] The PCR amplification reaction system is as follows: 1 μL cDNA, 1 μL KOD high-fidelity enzyme, 1 μL primer ZmCML11-F, 1 μL primer ZmCML11-R, 5 μL buffer, 10 μL dNTP mix, and sterile water to a final volume of 50 μL.

[0068] The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 50℃ annealing for 45 s, 72℃ extension for 60 s, for a total of 35 cycles, followed by 72℃ extension for 7 min.

[0069] The nucleotide sequence of primer 3301-ZmCML11-F is shown in SEQ ID NO. 7;

[0070] The nucleotide sequence of primer 3301-ZmCML11-R is shown in SEQ ID NO. 8;

[0071] S103. The PCR products obtained in S102 were extracted and sequenced using a gel extraction kit.

[0072] Bioinformatics analysis of S2 and ZmCML11 genes:

[0073] The sequencing results in S103 were compared with the nucleotide sequence of the maize ZmCML11 gene using DNAMAN software. If the nucleotide sequences were the same, the cloning was successful. The PCR product described in the successfully cloned S102 is the ZmCML11 target gene fragment.

[0074] Construction of S3, maize ZmCML11 gene overexpression vector:

[0075] The pCambia3301 vector was digested with BamHI to obtain a large fragment of the pCambia3301 vector. The large fragment of the pCambia3301 vector and the target fragment of CML11 described in S102 were ligated by Infusion ligase at 50°C for 15 min to obtain the ligation product.

[0076] The ligation reaction system consisted of: 2 μL of pCambia3301 vector fragment, 3 μL of ZmCML11 target fragment, and 5 μL of Infusion ligase.

[0077] The ligation product was transformed into competent E. coli cells. The transformed bacterial solution was spread on LB solid medium containing kanamycin and cultured overnight at 37°C. Positive clones were selected for culture and colony PCR identification to obtain the maize ZmCML11 gene overexpression vector, namely pCambia3301-ZmCML11 plasmid.

[0078] Maize genetic transformation of S4 and ZmCML11 genes:

[0079] The pCambia3301-ZmCML11 plasmid obtained from S3 was used to transform Agrobacterium tumefaciens, which then infected the immature embryos of the maize inbred line CAL. After induction culture, co-culture, selection culture, differentiation culture, rooting culture and transplanting to seedlings, positive plants of the ZmCML11 transgene were obtained.

[0080] Identification of positive plants of S5 and ZmCML11 transgenics:

[0081] The positive plants of the ZmCML11 transgene obtained in S4 were subjected to ZmCML11 gene PCR detection. A band of the same size as that in the pCambia3301-ZmCML11 plasmid described in the positive control S3 was amplified. After sequencing, the sequence alignment was correct, indicating that it was a maize ZmCML11 gene overexpression plant.

[0082] The sequence information involved in this application is as follows.

[0083] SEQ ID NO. 1:

[0084] ATGGCGATCAAGAACATGACGGTGGCGGCGACGACGCGGTCGCTGGACGCGGACATGACGGTGGACGAGTTCAAGGAGTGGCTGCGGCGGTTCGACACGGACCAGGACGGGCGCATCAGCCGCGACGAGCTGCGGCGCGCTCTGCGTGC CATCCGCTCCCGGTTCACGGGGTGGAGGAGCAGGCAGGGCATCAGTTATGCCGACACAGACGGCGACGGCTACATCGACGACAGCGAGGTCGATGGCCTCATCGAGTTCGCGCAGAAGAACCTCGGCCTCAAGATCGTCGCCTACTGA;

[0085] SEQ ID NO. 2:

[0086] MAIKNMTVAATTRSLDADMTVDEFKEWLRRFDTDQDGRISRDELRRALRAIRSRFTGWRSRQGISYADTDGDGYIDDSEVDGLIEFAQKNLGLKIVAY*;

[0087] SEQ ID NO. 3: GTGGACGAGTTCAAGGAGTGG;

[0088] SEQ ID NO. 4:GATCTTGAGGCCGAGGTTCTTC;

[0089] SEQ ID NO. 5: AGCAGGCTTTGACTTTATGGCGATCAAGAACA;

[0090] SEQ ID NO. 6: TGGGTCTAGAGACTTTCAGTAGGCGACGATCTTGAGGCCGAG;

[0091] SEQ ID NO. 7:CGACTCTAGAGGATCCATGGCGATCAAGAACATGACGGTG;

[0092] SEQ ID NO. 8:TGCTCACCATGGATCCGTAGGCGACGATCTTGAGGCC;

[0093] SEQ ID NO. 9:CAGGCTGGACGCGGACATGACGG;

[0094] SEQ ID NO. 10:AACCCGTCATGTCCGCGTCCAGC;

[0095] SEQ ID NO. 11:CATGGAGGCCGAATTCATGGCGATCAAGAACATGACGGTG;

[0096] SEQ ID NO. 12:GCAGGTCGACGGATCCTCAGTAGGCGACGATCTTGAGGCC;

[0097] SEQ ID NO. 13:AATACCACTACAATGGATGATG;

[0098] SEQ ID NO. 14:GAGATGGTGCACGATGCACAGT;

[0099] SEQ ID NO. 15:ATCTGTCGACCTCGAGATGGCGGCGTCCGCGA;

[0100] SEQ ID NO. 16:CGAGCTCGATGGATCCCTAGCTGAGGTTCAGGCTGGAATCCAC;

[0101] SEQ ID NO. 17:CACGGGGGACGAGCTCGGTACCATGGCGATCAAGAACATGACGGTG;

[0102] SEQ ID NO. 18:ACGCGTACGAGATCTGGTCGACGTAGGCGACGATCTTGAGGCC;

[0103] SEQ ID NO. 19:TACGCGTCCCGGGGCGGTACC ATGGCGGCGTCCGCGA;

[0104] SEQ ID NO. 20:ACGAAAGCTCTGCAGGTCGAC CTAGCTGAGGTTCAGG;

[0105] SEQ ID NO. 21:CGCCACTAGTGGATCCATGGCGATCAAGAACATGACGG;

[0106] SEQ ID NO. 22:TCCATCCCGGGAGCGGTACCTCAGTAGGCGACGATCTTGAGG;

[0107] SEQ ID NO. 23:CGCCACTAGTGGATCCATGGCGGCGTCCGCGAGG;

[0108] SEQ ID NO. 24:TACATCCCGGGAGCGGTACCCTAGCTGAGGTTCAGGCTGGAATCC;

[0109] SEQ ID NO. 25:CCGCTGCTCATCAACAAGAAG;

[0110] SEQ ID NO. 26:CGAGAACCGGTCCCTCTCTA;

[0111] SEQ ID NO. 27:TAGAGGGCCTCCCTTCTGAC;

[0112] SEQ ID NO. 28:AGCTGCACATACGACTCCAC;

[0113] SEQ ID NO. 29:CTCGCCTTATTCATAACCACAC;

[0114] SEQ ID NO. 30:TGGCCATCTGTTGATCTATCTG;

[0115] SEQ ID NO. 31:TCTCCTCCTCCTTCTCCTCG;

[0116] SEQ ID NO. 32:TGAACAGGTCCGTCTTGTCG;

[0117] SEQ ID NO. 33:ACGAGGCCATCTACAGCTTC;

[0118] SEQ ID NO. 34:TTGGTGTAGTTGTCGAGGGATG;

[0119] SEQ ID NO. 35: TGTACGAGATGAACGAGCGG;

[0120] SEQ ID NO. 36: GGAAGGCGGCTTGTTTGTG;

[0121] SEQ ID NO. 37: GTCGCAAGACGTTCTACAC;

[0122] SEQ ID NO. 38: ATCGCGGCATTACCCAGATG;

[0123] SEQ ID NO. 39: CCTTCGACCAACTTTTCCCCT;

[0124] SEQ ID NO. 40: ATGATGGTGCCCTCGGAGATG.

[0125] The 220, Y1518, and B73 mentioned in this application are maize inbred lines.

[0126] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0127] The present invention will be further illustrated below with reference to the embodiments.

[0128] Example 1

[0129] To investigate genes inducing low-temperature germination during the radicle emergence stage of seed 220, embryos of 220 and Y1518 seeds germinated at room temperature (25℃) for 36 h and at low temperature (10℃) for 240 h (at which point the germination process of the seeds in both treatments was similar, both at the radicle emergence stage), as well as their dry seed embryos, were subjected to transcriptome sequencing with three biological replicates. Differentially expressed genes were analyzed among those germinated at room temperature, low temperature, and dry seeds, using padj < 0.05 and |log2FoldChange| > 1 as criteria, yielding a total of 17652 differentially expressed genes. Cluster analysis of these gene expression patterns revealed a class of genes (Class E, 716 genes in total). These genes were almost not expressed in the dry seed embryos of 220 and Y1518, but were significantly upregulated in embryos germinated at both room temperature and low temperature, indicating that they are genotype-independent seed germination-inducing genes. Figure 1 As shown in (a) above. Furthermore, it was found that in Y220, their expression levels during low-temperature germination were significantly higher than those during normal-temperature germination; while in Y1518, there was no significant difference in expression levels between the two conditions, as shown in (a). Figure 1As shown in (a), these genes may be associated with the low-temperature germination characteristics of 220.

[0130] Of these 716 genes, the gene with the highest expression level (TPM reaching 3590) encodes a calmodulin-like protein, named ZmCML11. Biological information for ZmCML11 was retrieved from a maize database; its transcript ID is Zm00001d005895. The nucleotide sequence of the maize ZmCML11 gene is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

[0131] Example 2

[0132] To investigate the expression differences of ZmCML11 in the germination process of inbred lines 220 and Y1518, the expression level of ZmCML11 was verified in the embryos of seeds of 220 and Y1518 at different germination stages under normal temperature and low temperature. The specific methods are as follows.

[0133] The maize 220 and Y1518 inbred lines were selected as experimental materials. RNA was extracted from embryos germinated at room temperature for 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h, and from embryos germinated at low temperature for 0 h, 3 d, 6 d, 9 d, 12 d, and 15 d, and then reverse transcribed into cDNA.

[0134] Table 1: Reaction system for qRT-PCR quantification experiment

[0135]

[0136] The PCR amplification reaction program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 15 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 40 cycles, followed by 72℃ extension for 7 min.

[0137] The qRT-PCR primers are designed as follows:

[0138] q-ZmCML11-F: Nucleotide sequence as shown in SEQ ID NO. 3;

[0139] q-ZmCML11-R: The nucleotide sequence is shown in SEQ ID NO. 4.

[0140] Using the above cDNA as a template, a quantitative fluorescence experiment was performed using primers q-ZmCML11-F and q-ZmCML11-R. The results showed that, as Figure 1As shown in (b), the expression level of ZmCML11 in 220 seeds at all germination stages was significantly higher than that in Y1518 seeds at the same stage, and its expression was induced by low temperature. This indicates that ZmCML11 positively regulates seed germination at low temperature, which may be the reason for the difference in low-temperature germination between 220 and Y1518 seeds.

[0141] Example 3

[0142] This example demonstrates the subcellular localization of ZmCML11 in maize protoplasts.

[0143] Construction of S1 and pMDC83-ZmCML11 vectors

[0144] S101. Using the maize B73 inbred line as experimental material, RNA was extracted from embryos germinated at room temperature for 24 h and reverse transcribed into cDNA. Adapter primers were designed to amplify the ZmCML11 fragment. The pGWC plasmid was digested with Ahd1, and the fragment and linearized plasmid were purified using the Tiangen Universal DNA Purification and Recovery Kit (DP214). The pMDC83-ZmCML11 vector was constructed and the plasmid was upsampled.

[0145] Table 2: PCR amplification reaction system

[0146]

[0147] The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 50℃ annealing for 45 s, 72℃ extension for 60 s, for a total of 35 cycles, followed by 72℃ extension for 7 min.

[0148] The PCR amplification primers are designed as follows:

[0149] pGWC-ZmCML11-F: Nucleotide sequence as shown in SEQ ID NO. 5;

[0150] pGWC-ZmCML11-R: The nucleotide sequence is shown in SEQ ID NO. 6.

[0151] Preparation of S102 and pGWC linearized carriers

[0152] The enzyme digestion system is as follows:

[0153] Table 3: pGWC digestion system

[0154]

[0155] Purify and recover the enzyme digestion products.

[0156] S103, Recombination reaction

[0157] The connection system is as follows:

[0158] Table 4: pGWC-ZmCML11 Connection System

[0159]

[0160] Incubate at 50°C for 15 minutes, then immediately cool before conversion.

[0161] S104, Transformation Culture

[0162] Transform E. coli competent cells, screen for positive clones, and extract plasmid DNA for later use.

[0163] Take 100 μL of competent E. coli DH5α cells and mix with 10 μL of enzyme digestion and ligation product, and incubate on ice for 30 min. Then, quickly place the mixture in a 42℃ constant temperature water bath for 90 s heat shock and incubate on ice for 2 min. Add 500 μL of LB liquid medium and mix well. Then, incubate at 37℃ and 200 rpm for 45 min to allow the cells to recover to normal growth. Finally, spread the bacterial solution evenly on LB solid medium plates containing chloramphenicol resistance. Invert the plates and incubate overnight at 37℃.

[0164] S105, colony PCR and sequencing

[0165] Twelve single clones were selected, streaked and numbered, pipette tips were inserted into a plate containing the PCR system, mixed, and then PCR and electrophoresis were performed. Finally, sequencing was used to confirm the sequence was correct.

[0166] S106, Plasmid Extraction

[0167] The plasmids were extracted using the Tiangen Plasmid Mini-Prep Kit (DP103).

[0168] S107, LR reaction

[0169] The LR connection system is as follows:

[0170] Table 5: LR Reaction System

[0171]

[0172] Incubate at 25°C for 5 h. After incubation, add 1 μL of 10× Proteinase K Solution, mix well, and incubate at 37°C for 10 min. Transform the above cloning reaction into 50 μL of DH5α competent bacteria. After successful sequencing, extract the plasmid.

[0173] S2. Protoplasts were extracted from B73 etiolated seedlings that had germinated at room temperature in the dark for 14 days.

[0174] S201. Sow 40 B73 seeds in a 1:1 mixture of potting soil and vermiculite. Incubate in the dark for 10-12 days, until the second leaf has fully unfolded. Subsequent protoplast preparation steps are carried out under dim light conditions.

[0175] S202, Enzymatic hydrolysis of the cell wall

[0176] Take the widest part from the middle of the second leaf and cut it into 1 mm thin strips. Place the strips in 20 mL of enzymatic hydrolysis solution, ensuring complete protection from light. Then, perform vacuum permeation to promote enzymatic hydrolysis. Vacuum treatment for 30 min, followed by enzymatic hydrolysis on a shaker at 40 rpm for 4 h.

[0177] S202, Protoplast Filtration

[0178] Add 20 mL of W5 buffer to release the protoplasts. Fold a 300-mesh filter cloth into four layers, place it in a funnel, and secure it with a paperclip. Then, transfer the funnel to a 50 mL sterile centrifuge tube, rinse with W5 buffer, and place on ice. Next, filter the enzyme digest into the centrifuge tube. Replace the filter cloth and filter again to thoroughly remove other impurities.

[0179] S203, Protoplast Collection

[0180] Centrifuge at 4℃ / 100 g for 2 min with zero acceleration / deceleration. After centrifugation, carefully remove the supernatant, add 20 mL of pre-chilled W5 buffer, mix gently, and centrifuge again. Repeat the washing step to purify the protoplasts, resuspend them in 20 mL of W5 buffer, and incubate on ice for 30 min. During this time, the protoplasts can be observed using an optical microscope. After incubation, discard the supernatant and add an appropriate amount of pre-chilled MMG buffer.

[0181] S204 and PEG-mediated transformation

[0182] Add 10 μg each of pGWC83-HSP31 and DEK36 plasmids (concentration not less than 1000 ng / μL) to a 2 mL sterile centrifuge tube. Then add 100 μL of protoplasts and mix gently. Next, add 110 μL of PEG solution, mix gently, and let stand for 18–25 min. After the reaction is complete, add 440 μL of W5 buffer to terminate the transformation. Centrifuge at 4 °C / 100 g, with acceleration / deceleration at 0 for 2 min, discard the supernatant, add 1 mL of W5 buffer, and mix gently. Centrifuge again, carefully aspirate the supernatant, and wash away the reaction solution. Finally, add 1 mL of WI buffer and mix gently.

[0183] S205, protoplast culture

[0184] Wrap the centrifuge tubes in aluminum foil and incubate them in the dark at 25°C for 18 hours.

[0185] S3. Observe protoplasts using an LSM880 or LSM900 laser confocal microscope.

[0186] The results show that, Figure 1 As shown in (c), the ZmCML11 protein is located in the cell nucleus and cell membrane.

[0187] Example 4

[0188] To determine the molecular function of ZmCML11 in cold-tolerant germination of maize, overexpression lines of ZmCML11 were constructed using CAL inbred lines as a background.

[0189] This embodiment describes the application of the maize ZmCML11 gene, where the gene is overexpressed in maize plants to improve cold tolerance. The method for overexpressing the maize ZmCML11 gene in maize plants is as follows:

[0190] Cloning of S1 and ZmCML11 genes

[0191] S101. Using the CML11 fragment obtained in Example 3 as a template, PCR amplification was performed using primers 3301-ZmCML11-F and 3301-ZmCML11-R to obtain the PCR product. The PCR amplification reaction system is as follows:

[0192] Table 6: PCR amplification reaction system

[0193]

[0194] The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 50℃ annealing for 45 s, 72℃ extension for 60 s, for a total of 35 cycles, followed by 72℃ extension for 7 min.

[0195] The nucleotide sequence of primer 3301-ZmCML11-F is shown in SEQ ID NO. 7;

[0196] The nucleotide sequence of the primer 3301-ZmCML11-R is shown in SEQ ID NO. 8.

[0197] S103. The PCR products obtained in S102 were extracted and sequenced using a gel extraction kit.

[0198] Construction of S2 and maize ZmCML11 gene overexpression vector

[0199] S201. Linearize the vector pCambia3301 by single-enzyme digestion with BamHI. The digestion system is as follows:

[0200] Table 7: Single Enzyme Digestion Vector System

[0201]

[0202] Enzymatic digestion at 37℃ for 3-4 h yielded linearized digested products.

[0203] S202, Fragment and Linearized Vector Connection

[0204] Table 8: Connection System

[0205]

[0206] The ligation reaction was carried out at 50°C for 15 min to obtain the ligation product.

[0207] The ligation product was transformed into competent E. coli cells. The transformed bacterial culture was spread on LB solid medium containing kanamycin and cultured overnight at 37°C. Positive clones were selected for culture and plaque PCR identification to obtain the maize ZmCML11 gene overexpression vector, namely pCambia3301-ZmCML11 plasmid.

[0208] Maize genetic transformation of S3 and ZmCML11 genes

[0209] The pCambia3301-ZmCML11 plasmid obtained from S2 was used to transform Agrobacterium tumefaciens, which then infected the immature embryos of the maize inbred line CAL. After induction culture, co-culture, selection culture, differentiation culture, rooting culture, and transplanting, positive ZmCML11 transgenic plants were obtained.

[0210] The specific method is as follows: First, select uniformly sized immature embryos of the maize inbred line CAL. Remove the embryos in a sterile operating table and induce incubation at 34℃ for 3 days in the dark; then place them in an incubator at 25℃ for 7 days in the dark. Remove the endogenous buds from the induced callus and inoculate it into a subculture medium, incubating in the dark at 25℃ for 7 days. Subculture twice for use in Agrobacterium transformation. Place the callus tissue in a sterile infection medium and wash twice; after the last wash, discard the liquid and add bacterial suspension (OD). 600 =0.4), and infect for 30 min; after Agrobacterium infection, the embryos were dried on sterile filter paper, transferred to the surface of the co-culture medium, with the hypocotyl end in contact with the medium (scutellum facing upwards), and cultured in the dark at 23℃ for 3 days. After screening twice, once complete regenerated seedlings had grown, they were transferred to rooting medium and rooted and strengthened at 28℃ with 16 h light. When the roots reached about 2 cm in length, the seedlings were hardened off and transplanted.

[0211] Identification of positive plants of S4 and ZmCML11 transgenics

[0212] Positive plants of the ZmCML11 transgene obtained in S3 were subjected to ZmCML11 gene PCR detection. A band of the same size as that in the pCambia3301-ZmCML11 plasmid described in the positive control S2 was amplified. After sequencing, the sequence alignment was correct, indicating that the plants were overexpressing the maize ZmCML11 gene (e.g., Figure 3 (OE#1 and OE#2 in the text).

[0213] Example 5

[0214] To determine the molecular function of ZmCML11 in cold-tolerant germination of maize, knockout lines of ZmCML11 were constructed using CAL inbred lines as a background.

[0215] This embodiment describes a method for knocking out the ZmCML11 gene in maize plants.

[0216] The method for constructing the CRISPR / Cas9 knockout vector vk005-ZmCML11 is as follows:

[0217] Construction of S1 and VK005 gene knockout vectors

[0218] S101. A knockout target sequence fragment was found in the database of the National Center for Information Technology (NCBI), as shown below.

[0219] Target 1: The nucleotide sequence is GCTGGACGCGGACATGACGG (SEQ ID NO. 41).

[0220] The primers linked to the target sequence were designed as follows:

[0221] vk005-CML11-F: Nucleotide sequence as shown in SEQ ID NO. 9;

[0222] vk005-CML11-R: The nucleotide sequence is shown in SEQ ID NO. 10.

[0223] S102. First, the CRISPR-Cas9 site-directed mutagenesis technique was used to create a maize mutant with the gene loss of function. The sgRNA of ZmCML11 was designed using the online analysis software CRISPR-P2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ) and ligated into the VK005 vector.

[0224] The specific steps are as follows:

[0225] ① First, synthesize double-stranded DNA using PCR with one pair of primers for the vector;

[0226] The PCR reaction system is as follows: forward primer: 5 μL; reverse primer: 5 μL; H2O: 40 μL.

[0227] PCR program: pre-denaturation at 95℃ for 5 min, then quickly shut down the PCR instrument and allow it to cool naturally.

[0228] ② The VK005 plasmid was digested with BsqQ1 at 37℃ for 3-4 h and then purified.

[0229] The enzyme digestion reaction system is as follows:

[0230] Table 9: Enzyme digestion system of VK005 plasmid

[0231]

[0232] ③ Target site and linearized vector conjugation

[0233] The connection system is as follows:

[0234] Table 10: Target Carrier Connection System

[0235]

[0236] ④ Transform E. coli competent cells, screen for positive clones, and extract plasmid DNA for later use.

[0237] Take 100 μL of competent E. coli DH5α cells and mix with 10 μL of enzyme digestion and ligation product, then incubate on ice for 30 min. Immediately afterwards, place in a 42°C water bath for heat shock for 90 s, followed by an ice bath for 2 min. Add 500 μL of LB liquid medium and mix well. Then, incubate at 37°C and 200 rpm for 45 min to allow the cells to return to normal growth. Finally, evenly spread the bacterial culture onto LB solid medium plates containing kanamycin resistance, invert the plates, and incubate overnight at 37°C. Positive clones are then screened and sent to the company for sequencing.

[0238] ⑤ Agrobacterium-mediated transformation

[0239] Plasmid DNA was extracted. It was transformed into Agrobacterium and colony PCR was performed for verification. The bacteria were then preserved at -20°C. The CRISPR / Cas9 knockout vector pVK005-ZmCML11 was used to transform CAL embryos, using the same method as in Example 4 for transforming CAL embryos with the pCambia3301-ZmCML11 plasmid. The gene-editing knockout mutant zmcml11-ko-1 was finally obtained. Figure 2 crispr#1) and zmcml11-ko-2 ( Figure 2 (crispr#2 in the middle).

[0240] Example 6

[0241] To confirm the effect of ZmCML11 overexpression on seed germination at low temperatures, the following experiment was conducted.

[0242] S1. Select 30 plump and uniform seeds from each of the ZmCML11 OE mutants ZmCML11-OE-1, ZmCML11-OE-2, and their corresponding WT (CAL) materials harvested in the current season, forming one replicate, and set up 3 biological replicates. First, completely immerse the seeds in NaClO (sodium hypochlorite) solution for 10 minutes, and then rinse them with deionized water a total of 3 times to ensure that the NaClO on the seed surface is basically removed.

[0243] S2. The experimental method used is sand culture. To prepare for the experiment, fine sand was filtered to remove impurities and sterilized at 130℃. After cooling, the sand and deionized water were mixed evenly to maintain the sand moisture content at about 16%. The seeds were sown evenly on a 3 cm thick sand bed in a 5×6 pattern for each replicate, with the embryo side facing up. Finally, the germination box was sealed with a self-sealing bag to prevent moisture evaporation and placed in constant temperature incubators at 25℃ and 10℃ respectively.

[0244] S3. For seeds placed in a 25℃ incubator, observe the number of radicles protruding from the seed coat at 12-hour intervals. Radicles appear after 24 hours, and germination continues until all seeds have germinated at 72 hours. Record the germination morphology at 72 hours. Statistically analyze the germination rate, final germination rate, germination index (GI), and mean germination time (MGT) during the germination stage.

[0245] S4. For seeds placed in a 10℃ incubator, observe the number of radicles protruding from the seed coat at 24-hour intervals. Radicles appear after 5 days, and germination continues until all seeds have germinated by day 9. Record the germination morphology at day 7. Statistically analyze the germination rate, final germination rate, germination index (GI), and mean germination time (MGT) during the germination stage.

[0246] The results show that, Figure 3 As shown, there were no significant differences in GR, GI, and MGT between ZmCML11-OE-1 and OE2 mutant seeds under normal temperature conditions, but the germination rate under low temperature stress for 5-7 days was significantly higher than that of WT (CAL). There was no significant difference in the final germination rate between ZmCML11-OE-1 and OE2 mutants and WT (CAL). ZmCML11-OE-1 and OE2 mutants had higher GI and lower MGT than WT (CAL), proving that ZmCML11 positively regulates seed germination.

[0247] Example 7

[0248] This example is a low-temperature germination experiment of the gene-edited knockout mutants zmCML11-ko-1 and zmcml11-ko-2 from Example 5:

[0249] S1. Select 10 plump and uniform seeds from the ZmCML11 KO mutant zmCML11-ko-2 and the corresponding WT (CAL) material harvested in the current season as one replicate, and set up 3 biological replicates. First, completely immerse the seeds in NaCIO (sodium hypochlorite) solution for 10 minutes, and then rinse with deionized water a total of 3 times to ensure that the NaCIO on the seed surface is basically removed.

[0250] S2. The experimental method used is sand culture. To prepare for the experiment, fine sand is filtered to remove impurities and sterilized at 130℃. After cooling, the sand and deionized water are mixed evenly to keep the sand moisture content at about 16%. The seeds are sown evenly on a 3 cm thick sand bed in a 5×6 pattern for each replicate, with the embryo side facing up. Finally, the germination box is sealed with a self-sealing bag to prevent moisture evaporation and placed in a constant temperature incubator at 25℃ and 10℃.

[0251] S3. For seeds placed in a 25℃ incubator, observe the number of radicles protruding from the seed coat at 12-hour intervals. Radicles appear after 24 hours, and germination continues until all seeds have germinated at 72 hours. Record the germination morphology at 72 hours. Statistically analyze the germination rate, final germination rate, germination index (GI), and mean germination time (MGT) during the germination stage.

[0252] S4. For seeds placed in a 10℃ incubator, observe the number of radicles protruding from the seed coat at 24-hour intervals. Radicles appear after 6 days, and germination continues until all seeds have germinated by 10 days. Record the germination morphology at 7 days. Statistically analyze the germination rate, final germination rate, germination index (GI), and mean germination time (MGT) during the germination stage.

[0253] The results show that, Figure 2 As shown, the zmcml11-ko-2 mutant seeds showed no significant differences in GR, GI, and MGT under normal temperature conditions, but the germination rate under low temperature stress for 7 days was significantly lower than that of WT (CAL). There was no significant difference in the final germination rate between the zmcml11-ko-2 mutant and WT (CAL). The zmcml11-ko-2 mutant had a lower GI and a higher MGT than WT (CAL), proving that ZmCML11 positively regulates seed germination at low temperatures.

[0254] Example 8

[0255] Calmodulin-like protein ZmCML11, because it contains only the EF domain, can bind to Ca... 2+ It binds, but must interact with other proteins to transfer Ca. 2+Signal transduction, thereby exerting corresponding biological functions. Therefore, in order to elucidate the molecular mechanism of ZmCML11 in low-temperature germination of maize, a yeast dual-hybrid screening library experiment was conducted to identify interacting proteins that interact with it to exert downstream functions. The specific methods are as follows.

[0256] Construction of S1 and pGBKT7 vectors and yeast transformation

[0257] S101. Using the ZmCML11 fragment obtained in Example 3 as a template, PCR amplification was performed using primers BD-ZmCML11-F and BD-ZmCML11-R to obtain the PCR product. The PCR amplification reaction system was as follows:

[0258] Table 11: PCR amplification reaction system

[0259]

[0260] The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 50℃ annealing for 45 s, 72℃ extension for 60 s, for a total of 35 cycles, followed by 72℃ extension for 7 min.

[0261] The nucleotide sequence of the primer BD-ZmCML11-F is shown in SEQ ID NO. 11;

[0262] The nucleotide sequence of the primer BD-ZmCML11-R is shown in SEQ ID NO. 12;

[0263] S102. The pGBKT7 plasmid was digested with BamH1 and EcoR1 at 37℃ for 3-4 h, and then purified. The digestion reaction system was as follows:

[0264] Table 12: Enzyme digestion system of pGBKT7 plasmid

[0265]

[0266] S103, Target site and linearized vector connection

[0267] The connection system is as follows:

[0268] Table 13: Target Carrier Connection System

[0269]

[0270] S104. Transform E. coli competent cells, screen for positive clones, and extract plasmid DNA for later use.

[0271] Take 100 μL of competent E. coli DH5α cells and mix with 10 μL of enzyme digestion and ligation product, then incubate on ice for 30 min. Immediately afterwards, place in a 42°C water bath for heat shock for 90 s, followed by an ice bath for 2 min. Add 500 μL of LB liquid medium and mix well. Then, incubate at 37°C and 200 rpm for 45 min to allow the cells to return to normal growth. Finally, evenly spread the bacterial culture onto LB solid medium plates containing kanamycin resistance, invert the plates, and incubate overnight at 37°C. Positive clones are then screened and sent to the company for sequencing.

[0272] S105, yeast transformation

[0273] Take 100 μL of competent yeast cells Y2HGold, mix with 5 μg pGBKT7-CML11 plasmid, 10 µL Carrier DNA, and 500 µL PEG / LiAc, incubate at 30℃ for 30 min, and invert 6-8 times at 15 min to mix; then place in a 42℃ constant temperature water bath for heat shock for 15 min, and invert 6-8 times at 7.5 min to mix; centrifuge at 10,000 rpm for 30 s and discard the supernatant, resuspend in 400 µL ddH2O, centrifuge for 30 s and discard the supernatant; resuspend in 50 µL ddH2O, plate on SD-Trp plates, and incubate at 28-30℃ for 48-96 h.

[0274] S2, Yeast Screening Library

[0275] S201, Self-activation detection

[0276] Take 100 μL of competent yeast cells (Y2HGold) and mix with 5 μg pGBKT7-CML11 plasmid, 5 µg pGADT7 plasmid, 10 µL carrier DNA, and 500 µL PEG / LiAc. Incubate at 30℃ for 30 min, inverting 6-8 times at 15 min to mix thoroughly. Then place in a 42℃ constant temperature water bath for heat shock for 15 min, inverting 6-8 times at 7.5 min to mix thoroughly. Centrifuge at 10,000 rpm for 30 s, discard the supernatant, resuspend in 400 µL ddH2O, centrifuge for 30 s, discard the supernatant, resuspend in 50 µL ddH2O, plate on SD / -Leu / -Trp plates, and incubate at 28-30℃ for 48-96 h. Pick a fresh single colony and resuspend in 1 mL of 0.9% sodium chloride solution. OD 600 Adjust the concentration to 0.002, take 100 μL and spread it on SD / -Leu / -Trp and SD / -His / -Leu / -Trp solid culture medium; place the culture dish in a 30℃ constant temperature incubator and incubate for 3~5 days until clones grow, and observe the yeast growth trend.

[0277] S202, Toxicity Testing

[0278] Fresh yeast single colonies carrying pGBKT7-CML11 and pGBKT7 were picked separately, and OD was measured. 600 Adjust the concentration to 0.002, then take 100 μL and spread it onto SD / -Trp solid medium. Place the culture dish in a 30℃ incubator and incubate for 3-5 days until clones grow. Observe the yeast growth trend.

[0279] S203, mating method screening library

[0280] Take a fresh 2-3 mm single colony carrying pGBKT7-CML11 and inoculate it into an Erlenmeyer flask containing 50 mL of SD / -Trp liquid medium; incubate at 30°C with shaking at 200 rpm until the OD value is 0.8; centrifuge at 3000 rpm for 5 min, discard the supernatant, and resuspend the pellet in 4-5 mL of SD / -Trp liquid medium to achieve a cell concentration greater than 1×10⁻⁶. 8 / mL; Transfer 1 mL of Y187 library yeast strain and 4-5 mL of Y2H-pGBKT7-CML11 strain to a sterile 2 L Erlenmeyer flask, add 45 mL of 2×YPDA liquid medium; incubate at 30℃ with gentle shaking at 30-50 rpm for 20-24 h; centrifuge at 3000 rpm for 10 min, while rinsing the 2 L Erlenmeyer flask with 50 mL of 0.5×YPDA, and resuspend the centrifuged cells; centrifuge at 3000 rpm for 10 min, remove the supernatant, resuspend the precipitate with 10 mL of 0.5×YPDA, and spread 200 μL each time onto SD / -Ade / -His / -Leu / -Trp solid medium (Φ150 mm petri dish), and incubate at 30℃ for 3-5 days.

[0281] S204. Screening for positive clones

[0282] The obtained single clones were streaked on SD / -Ade / -His / -Leu / -Trp solid selective medium, and colony PCR and sequencing were performed using pGADT7 universal primers. The sequencing results of positive clones were analyzed online using the BLAST sequence alignment tool.

[0283] The universal primers for pGADT7 are as follows:

[0284] 5AD: The nucleotide sequence is shown in SEQ ID NO. 13;

[0285] 3AD: The nucleotide sequence is shown in SEQ ID NO. 14.

[0286] Zmzim23, a key inhibitor of the jasmonic acid signaling pathway, was obtained from yeast positive colonies. To verify the authenticity of the interaction, a yeast two-hybrid experiment was performed on ZmCML11 and Zmzim23.

[0287] S3, yeast hybrid

[0288] S301, pGADT7 plasmid ligation

[0289] Using the cDNA obtained in Example 3 as a template, PCR amplification was performed using primers AD-Zmzim23-F and AD-Zmzim23-R to obtain PCR products. The PCR amplification reaction system was as follows:

[0290] Table 14: PCR amplification reaction system

[0291]

[0292] The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 50℃ annealing for 45 s, 72℃ extension for 60 s, for a total of 35 cycles, followed by 72℃ extension for 7 min.

[0293] The nucleotide sequence of the primer AD-Zmzim23-F is shown in SEQ ID NO. 15;

[0294] The nucleotide sequence of the primer AD-Zmzim23-R is shown in SEQ ID NO. 16.

[0295] S302. The pGADT7 plasmid was digested with BamH1 and EcoR1 at 37℃ for 3-4 h, and then purified. The digestion reaction system was as follows:

[0296] Table 15: Enzyme digestion system of pGADT7 plasmid

[0297]

[0298] S303, Target site and linearized vector connection

[0299] The connection system is as follows:

[0300] Table 16: Target Carrier Connection System

[0301]

[0302] S304. Transform E. coli competent cells, screen for positive clones, and extract plasmid DNA for later use.

[0303] Take 100 μL of competent E. coli DH5α cells and mix with 10 μL of enzyme digestion and ligation product, then incubate on ice for 30 min. Immediately afterwards, place in a 42°C water bath for heat shock for 90 s, followed by an ice bath for 2 min. Add 500 μL of LB liquid medium and mix well. Then, incubate at 37°C and 200 rpm for 45 min to allow the cells to return to normal growth. Finally, evenly spread the bacterial culture onto LB solid medium plates containing ampicillin. Invert the plates and incubate overnight at 37°C. Positive clones are then screened and sent to the company for sequencing.

[0304] S305, plasmid co-transformed yeast

[0305] Take 100 µL of AH109 competent cells thawed on ice, and add 5 µg each of pre-chilled plasmids pGBKT7-ZmCML11 and pGADT7-Zmzim23, 10 µL of carrier DNA, and 500 µL of PEG / LiAc. Mix well by pipetting several times. Using the same system, add 5 µg each of pGBKT7-p53 and pGADT7-T, pGBKT7-Lam and pGADT7-T, pGBKT7-ZmCML11 and pGADT7, and pGBKT7 and pGADT7-Zmzim23 plasmids to four other competent cell lines as positive controls, negative controls, and self-activation controls, respectively. Incubate at 30°C for 30 min (twisting 6-8 times at 15 min to mix); incubate at 42°C for 15 min (twisting 6-8 times at 7.5 min to mix); centrifuge at 10,000 rpm for 30 minutes. Discard the supernatant, resuspend in 400 µL of ddH2O, centrifuge for 30 s and discard the supernatant; resuspend in 50 µL of ddH2O, plate on SD / -Leu / -Trp solid medium, and incubate at 28-30℃ for 48-96 h; pick fresh single colonies (2-3 mm) from each sample and resuspend in 1 mL of sterile ddH2O water, OD 600 Adjust to 0.2; dilute with ddH2O 10 times, 100 times, and 1000 times in sequence, and spot 10 µL of bacterial suspension onto SD / -Leu / -Trp and SD / -Leu / -Trp / -His / -Ade plates in the order of experimental group first and then control group, and incubate at 30℃ for 3~5 days.

[0306] Experimental results demonstrate that ZmCML11 and Zmzim23 proteins interact in yeast. ZmCML11 may affect the low-temperature germination of maize seeds through the jasmonic acid pathway.

[0307] Example 9

[0308] To further demonstrate the interaction between ZmCML11 and Zmzim23 proteins, their interaction relationship was further verified using tobacco luciferase complementation imaging technology. The specific methods are as follows.

[0309] Construction of S1, nLUC-ZmCML11 and cLUC-Zmzim23 vectors and their transformation with Agrobacterium tumefaciens

[0310] S101, Plasmid Ligation

[0311] Using the ZmCML11 and Zmzim23 fragments obtained in Example 8 as templates, PCR amplification was performed using primers nLUC-ZmCML11-F and nLUC-ZmCML11-R, as well as cLUC-Zmzim23-F and cLUC-Zmzim23-R, to obtain PCR products. The reaction system for the PCR amplification was as follows:

[0312] Table 17: PCR amplification reaction system

[0313]

[0314] Table 18: PCR amplification reaction system

[0315]

[0316] The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 50℃ annealing for 45 s, 72℃ extension for 60 s, for a total of 35 cycles, followed by 72℃ extension for 7 min.

[0317] The nucleotide sequence of the primer nLUC-ZmCML11-F is shown in SEQ ID NO. 17;

[0318] The nucleotide sequence of the primer nLUC-ZmCML11-R is shown in SEQ ID NO. 18;

[0319] The nucleotide sequence of the primer cLUC-Zmzim23-F is shown in SEQ ID NO. 19;

[0320] The nucleotide sequence of the primer cLUC-Zmzim23-R is shown in SEQ ID NO. 20;

[0321] S102. The nLUC and cLUC plasmids were digested with Kpn1 and Sal1 at 37℃ for 3-4 h and then purified.

[0322] The enzyme digestion reaction system is as follows:

[0323] Table 19: Enzyme digestion systems for nLUC and cLUC plasmids

[0324]

[0325] S103, Target site and linearized vector connection

[0326] The connection system is as follows:

[0327] Table 20: Target Carrier Connection System

[0328]

[0329] S104. Transform E. coli competent cells and screen for positive clones.

[0330] Take 100 μL of competent E. coli DH5α cells and mix with 10 μL of enzyme digestion and ligation product, incubate on ice for 30 min; then quickly place in a 42℃ water bath for heat shock for 90 s, followed by an ice bath for 2 min; add 500 μL of LB liquid medium and mix well; then incubate at 37℃ and 200 rpm for 45 min to allow the cells to recover to normal growth; finally, evenly spread the bacterial culture on LB solid medium plates containing kanamycin resistance; invert the plates and incubate overnight at 37℃. Positive clones are then screened and sent to the company for sequencing.

[0331] S105. Plasmids with correct sequencing were transformed into Agrobacterium tumefaciens and used to transform tobacco for observation.

[0332] The results are as follows Figure 4 (b) shows that ZmCML11 and Zmzim23 proteins interact with each other in tobacco.

[0333] Example 10

[0334] To further demonstrate the interaction between ZmCML11 and Zmzim23 proteins, their interaction relationship was verified in vivo in plants using bimolecular fluorescence complementation technology. The specific methods are as follows.

[0335] Construction of S1, YNE-ZmCML11 and YCE-Zmzim23 vectors and their transformation with Agrobacterium tumefaciens

[0336] S101, Plasmid Ligation

[0337] Using the ZmCML11 and Zmzim23 fragments obtained in Example 8 as templates, PCR amplification was performed using primers YNE-ZmCML11-F and YNE-ZmCML11-R, and YCE-Zmzim23-F and YCE-Zmzim23-R, to obtain PCR products. The reaction system for the PCR amplification was as follows:

[0338] Table 21: PCR amplification reaction system

[0339]

[0340] Table 22: PCR amplification reaction system

[0341]

[0342] The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 50℃ annealing for 45 s, 72℃ extension for 60 s, for a total of 35 cycles, followed by 72℃ extension for 7 min.

[0343] The nucleotide sequence of the primer YNE-ZmCML11-F is shown in SEQ ID NO. 21;

[0344] The nucleotide sequence of the primer YNE-ZmCML11-R is shown in SEQ ID NO. 22;

[0345] The nucleotide sequence of the primer YCE - Zmzim23-F is shown in SEQ ID NO. 23;

[0346] The nucleotide sequence of the primer YCE - Zmzim23-R is shown in SEQ ID NO. 24.

[0347] S102. YNE and YCE plasmids were digested with Kpn1 and BamH1 at 37℃ for 3-4 h, and then purified. The digestion reaction system was as follows:

[0348] Table 23: Enzyme digestion systems for YNE and YCE plasmids

[0349]

[0350] S103, Target site and linearized vector connection

[0351] The connection system is as follows:

[0352] Table 24: Target Carrier Connection System

[0353]

[0354] S104. Transform E. coli competent cells and screen for positive clones.

[0355] Take 100 μL of competent E. coli DH5α cells and mix with 10 μL of enzyme digestion and ligation product, incubate on ice for 30 min; then quickly place in a 42℃ water bath for heat shock for 90 s, followed by an ice bath for 2 min; add 500 μL of LB liquid medium and mix well; then incubate at 37℃ and 200 rpm for 45 min to allow the cells to recover to normal growth; finally, evenly spread the bacterial culture on LB solid medium plates containing kanamycin resistance; invert the plates and incubate overnight at 37℃. Positive clones are then screened and sent to the company for sequencing.

[0356] S105. Transform the correctly sequenced plasmid into Agrobacterium, and observe the tobacco leaves using an LSM880 or LSM900 laser confocal microscope.

[0357] The results are as follows Figure 4 (c) shows that ZmCML11 and Zmzim23 proteins interact in real protein interactions within the tobacco cell nucleus. These interaction experiments indicate that ZmCML11 interacts with Zmzim23 in the nucleus to influence the jasmonic acid signaling pathway, thereby regulating low-temperature germination of maize seeds.

[0358] Example 11

[0359] To investigate the effects of ZmCML11 on the jasmonic acid (JA) signaling pathway during low-temperature germination, key genes in the JA signaling pathway and downstream marker genes affected by it were explored. Studies have shown that in the model plant Arabidopsis thaliana, lipoxygenase (LOX), allylene oxide cyclase (AOC), allylene oxide synthase (AOS), and OPDA reductase (OPR3) are important catalytic enzymes in the biosynthesis of endogenous JA. Furthermore, due to the feedback regulation mechanism of JA, these genes are often considered downstream marker genes in the JA pathway. Therefore, homologous genes of these genes in maize were identified, and their expression levels in WT and knockout lines were compared. The specific experimental procedure is as follows.

[0360] S1. The zmcml11 knockout line obtained in Example 5 was selected as the experimental material. RNA was extracted from the embryos of the knockout line and wild-type radicles during the radicle protrusion stage and reverse transcribed into cDNA.

[0361] S2. qRT-PCR experiments were performed on the Arabidopsis jasmonic acid signaling pathway marker genes in maize to determine their expression levels. Using the cDNA obtained in S1 as a template, primers q-lox10-F and q-lox10-R, q-lox11-F and q-lox11-R, q-aos3-F and q-aos3-R, q-aos4-F and q-aos4-R, q-aoc1-F and q-aoc1-R, q-aoc2-F and q-aoc2-R, q-opr7-F and q-opr7-R, and q-opr8-F and q-opr8-R were used to determine the expression levels of the above genes in the zmcml11 knockout line and its wild type.

[0362] The nucleotide sequence of the primer q-lox10-F is shown in SEQ ID NO. 25;

[0363] The nucleotide sequence of the primer q-lox10-R is shown in SEQ ID NO. 26;

[0364] The nucleotide sequence of the primer q-lox11-F is shown in SEQ ID NO. 27;

[0365] The nucleotide sequence of the primer q-lox11-R is shown in SEQ ID NO. 28;

[0366] The nucleotide sequence of the primer q-aos3-F is shown in SEQ ID NO. 29;

[0367] The nucleotide sequence of the primer q-aos3-R is shown in SEQ ID NO. 30;

[0368] The nucleotide sequence of the primer q-aos4-F is shown in SEQ ID NO. 31;

[0369] The nucleotide sequence of the primer q-aos4-R is shown in SEQ ID NO. 32;

[0370] The nucleotide sequence of the primer q-aoc1-F is shown in SEQ ID NO. 33;

[0371] The nucleotide sequence of the primer q-aoc1-R is shown in SEQ ID NO. 34;

[0372] The nucleotide sequence of the primer q-aoc2-F is shown in SEQ ID NO. 35;

[0373] The nucleotide sequence of the primer q-aoc2-R is shown in SEQ ID NO. 36;

[0374] The nucleotide sequence of the primer q-opr7-F is shown in SEQ ID NO. 37;

[0375] The nucleotide sequence of the primer q-opr7-R is shown in SEQ ID NO. 38;

[0376] The nucleotide sequence of the primer q-opr8-F is shown in SEQ ID NO. 39;

[0377] The nucleotide sequence of the primer q-opr8-R is shown in SEQ ID NO. 40;

[0378] The reaction system for the qRT-PCR quantitative experiment is as follows:

[0379] Table 25: Reaction system for qRT-PCR quantification experiment

[0380]

[0381] The PCR amplification reaction program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 15 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 40 cycles, followed by 72℃ extension for 7 min.

[0382] Experimental results are as follows Figure 5 As shown in (a) to (d), the expression levels of most marker genes in the knockout lines were lower than those in the wild-type. This indicates that the jasmonic acid signaling pathway is suppressed in the knockout lines compared to the wild-type. This experiment further demonstrates that ZmCML11 regulates seed germination by affecting the jasmonic acid pathway at low temperatures.

[0383] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of the ZmCML11 gene or its encoded protein in any of the following: (i) Prediction of maize traits; (ii) Maize breeding; (iii) Maize genetic improvement; The nucleotide sequence of the ZmCML11 gene has: (1) The nucleotide sequence as shown in SEQ ID NO: 1; or (2) A nucleotide sequence obtained by substitution, deletion or addition of one or more bases to the nucleotide sequence shown in (1), and whose function is the same as or similar to that of (1); or (3) A nucleotide sequence that is at least 90% homologous to the nucleotide sequence shown in (1) or (2); The amino acid sequence of the protein has: (4) The amino acid sequence as shown in SEQ ID NO: 2; or (5) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (4), and whose function is the same as or similar to that of (4); or (6) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (4) or (5); The number of items is 2 to 50.

2. The application as described in claim 1, characterized in that, The maize trait prediction includes: (i) the ability to predict low-temperature germination in maize; and / or (ii) Predicting the cold resistance of maize; The maize breeding program includes: (iii) Screening for maize varieties with low-temperature germination ability; and / or (iv) Select corn varieties with higher cold resistance; The genetic improvement of maize includes: (v) The ability to induce low-temperature germination in maize; and / or (vi) Improve the cold resistance of corn.

3. A method for predicting maize traits, characterized in that, include: Prediction based on the ZmCML11 gene or its encoded protein in the application described in claim 1.

4. The method for predicting maize traits as described in claim 3, characterized in that, The criteria for determination include: higher expression levels of the ZmCML11 gene or its encoded protein indicate higher low-temperature germination rate and / or higher cold resistance in maize.

5. A method for breeding maize, characterized in that, include: Based on the ZmCML11 gene or the protein encoded by it described in the application of claim 1, selection and breeding were carried out.

6. The maize breeding method as described in claim 5, characterized in that, include: Corn was screened based on the expression levels of the ZmCML11 gene or its encoded protein to obtain corn with higher low-temperature germination rates and / or higher cold resistance.

7. A method for genetic improvement of maize, characterized in that, include: Genetic modification is carried out based on the ZmCML11 gene or the protein it encodes in the application described in claim 1.

8. The method for genetic improvement of maize as described in claim 7, characterized in that, include: Overexpression of the ZmCML11 gene or its encoded protein as described in claim 1 yields maize with higher low-temperature germination rate and / or higher cold resistance.

9. The method for genetic improvement of maize as described in claim 8, characterized in that, The ZmCML11 gene or its encoded protein interacts with the Zmzim23 protein to affect the jasmonic acid signaling pathway, thereby regulating low-temperature germination and / or cold resistance of maize seeds.

10. A biological material used for maize trait prediction, maize breeding, or maize genetic improvement, characterized in that, include: (i) The amplification primers for the ZmCML11 gene as described in claim 1; and / or (ii) A capture antibody for the protein described in claim 1; and / or (iii) Expression cassettes, expression vectors and / or recombinant host cells of the ZmCML11 gene or the protein encoded by it as described in claim 1.

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